The present invention provides methods for simultaneously assessing microbial phosphoglucose isomerase, ketol-isomerase and glucosamine-6-phosphate acetyltransferase activities, by measuring the production of Coenzyme A (CoA). The present invention finds use in the isolation of new classes of antifungal drugs, wherein the compounds have the ability to inhibit fungal glucose utilization.
During the last three decades there has been a dramatic increase in the frequency of fungal infections, especially disseminated systemic mycoses in immunocompromised patients (Cox and Perfect, Curr. Opin. Infect. Dis. 6:422-426 [1993]; and Fox, ASM News 59:515-518 [1993]). Human pathogenic fungi of particular importance include: Candida sp. (C. albicans, C. glabrata, C. krusei and C. parapsilosis), Aspergillus fumigatus, and Cryptococcus neoformans. C. albicans and A. fumigatus cause most opportunistic mycoses. At present, treatments for fungal infections are limited to few options. Amphotericin B (a polyene) is fungicidal, but is toxic to humans. Azoles (fluconazole, itraconazole, and others) are safer than amphotericin B, but are only fungistatic. In addition, resistance to azoles has become a major clinical concern. Some azoles (Sheehan, Clin. Microbiol. Rev. 12:40-79 [1999]) and a new class of (1,3)xcex2-glucan synthase inhibitors, echinocandins (Denning, J. Antimicrob. Chemother. 40:611-614 [1997]), are now in clinical and pre-clinical trials. Recently, the FDA approved caspofungin, a (1,3)xcex2-glucan synthase inhibitor, as a xe2x80x9csalvage treatmentxe2x80x9d for aspergillosis. In spite of the new azoles and the (1,3)xcex2-glucan synthase inhibitors, new classes of antifungal drugs are needed for therapy of infections caused by drug-resistant mutants (and species) or for preventing the emergence of drug-resistant mutants.
Fungal Infections and Drug Resistance
Fungal organisms have become increasingly significant pathogens in immunocompromised patients, especially those who because of cancer, organ transplantation, chemotherapy, pregnancy, age, diabetes, complications following extensive surgery, and various immune system dysfunctions, are at risk of experiencing life-threatening diseases caused by microorganisms which do not ordinarily pose a threat to normal, immunocompetent people. Other risk factors for deeply invasive fungal infections include protracted treatment using broad spectrum antimicrobials, corticosteroids, and vascular catheters.
Indeed, immunocompromised patients provide a significant challenge to modern health care delivery. For example, fungal infections have become one of the leading factors contributing to morbidity and mortality in cancer patients, and fungi account for 4-12% of nosocomial pathogens in leukemia patients (Anaissie, Clin. Infect. Dis., 14[Suppl. 1]:S43 [1992]). The incidence of nosocomial bloodstream infections with fungi such as Candida spp. (xe2x80x9ccandidemiaxe2x80x9d) has increased in recent years and has been reported to account for 5.6% of all primary bloodstream infections. There are an estimated 200,000 patients/year who acquire nosocomial fungal infections, with bloodstream infections having a mean mortality rate of 55% (See e.g., Beck-Sague et al., J. Infect. Dis., 167:1247 [1993]; and the Centers for Disease Control website at www.cdc.gov/ncidod/publications/brochures/hip.html). Fungal infections in non-humans, such as livestock, is also of significant health and economic concern. The most common fungal pathogens in humans are the opportunistic yeast, Candida albicans and the filamentous mold, Aspergillus fumigatus (See, Bow, Br. J. Haematol., 101:1 [1998]; and Warnock, J. Antimicrob. Chemother., 41:95 [1998]).
C. albicans is the most common fungal pathogen in humans, with other Candida species becoming increasingly important in fungal disease in humans and other animals (See, Walsh and Dixon, xe2x80x9cSpectrum of Mycoses,xe2x80x9d in Baron [ed.], Medical Microbiology, 4th ed, University of Texas Medical Branch, Galveston, Tex. [1996], pp. 919-925). Approximately 200 Candida species are recognized, with approximately seven of those species isolated with significant frequency from human specimens (See, Warren and Hazen, Ch. 95, pp. 1184-1199, xe2x80x9cCandida, Cryptococcus, and Other Yeasts of Medical Importance,xe2x80x9d in Murray et al., [eds.], Manual of Clinical Microbiology, 7th ed., ASM Press, Washington, D.C. [1999]; and Mitchell, in Zinsser Microbiology, Joklik et al., [eds], Appleton, Century-Crofts, Norwalk, Conn., pp. 1183-1190 [1984]).
The clinical manifestations of Candida infections and disease are many and varied, as Candida species are known to invade most organ systems of the body. Superficial candidiasis may involve the epidermal and mucosal surfaces (e.g., the oral cavity, pharynx, esophagus, intestines, urinary bladder, and vagina). In deep candidiasis, the gastrointestinal tract and intravascular catheters are the two major portals of entry, with the kidneys, liver, spleen, brain, eyes, heart, and other tissues being the major sites involved.
The major difficulties in treating Candida infections are encountered in cases of systemic disease. Chronic mucocutaneous, pulmonary candidiasis, endocarditis, and fungemia must be diagnosed early in order to avoid fatality. The incidence of candidiasis in certain patient populations is striking. Up to 30% of leukemia patients acquire systemic candidiasis (Anaissie, Clin. Infect. Dis., 14[Suppl. 1]:S43 [1992]). This is of great significance, as some reports indicate that the fatality rate for disseminated candidiasis in cancer patients is as high as 80% (Meunier, et al., Clin. Infect. Dis., 14[Suppl. 1]:S120 [1992]).
Aspergillus species are the second most common isolate, after Candida species, in patients with positive fungal cultures (See, Sigler and Kennedy, Ch. 97, xe2x80x9cAspergillus, Fusarium, and Other Opportunistic Moniliaceous Fungi,xe2x80x9d in Murray et al., (eds), Manual of Clinical Microbiology, 7th ed., ASM Press, Washington, D.C. [1999], pp. 1213-1241; and Goodwin et al., J. Med. Vet. Mycol., 30:153 [1992]). A large number of species of the genus Aspergillus have clinical relevance, although A. fumigatus, A. niger and A. flavus are most commonly isolated. Of these isolates, A. fumigatus is the most common human pathogen. Three main types of disease have been associated with A. fumigatus, namely allergic asthma, aspergilloma, and invasive aspergillosis (See e.g., Lortholary et al., Amer. J. Med., 95:177-187 [1993]).
Allergic pulmonary asthma due to A. fumigatus exposure affects an estimated 50,000 individuals in the United States alone. Aspergillomas are formed when fungal spores germinate in situ in tissue such as the lungs and form fungus balls. There is typically no invasion of underlying tissues, and in most cases treatment involves the simple surgical removal of the aspergilloma. However, invasive aspergillosis involves the invasion of host tissues, and is most commonly observed in patients with predisposing conditions (e.g., immunosuppressive drugs, neutropenia, chemotherapy, AIDS). Transplant (e.g., bone marrow or organ) and chemotherapy patients are at the greatest risk for this form of aspergillosis (See e.g., Denning et al., New Eng. J. Med., 324:654-662 [1992]; and Miller et al., Chest 105:37-44 [1994]). The prognosis for patients with invasive aspergillosis is particularly grave, with mortality rates greater than 50% (See e.g., Polis et al., xe2x80x9cFungal Infections in Patients with the Acquired Immunodeficiency Syndrome,xe2x80x9d in DeVita et al. (eds), AIDS: Biology, Diagnosis, Treatment, and Prevention, 4th ed., Lippincott-Raven, [1997]), due to the lack of a rapid diagnostic method to confirm A. fumigatus infection, and the lack of safe antifungal drugs.
The development of effective antifungal agents has lagged behind that of antibacterial agents. Fungi, like humans, are eukaryotic. Thus, most agents that have antimicrobial activity towards fungi are also toxic to humans (i.e., due to non-selective toxicity). Four general groups of antifungals have been developed; these are the polyenes, the azoles, the allylamines/morpholines, and the antimetabolites. Despite the identification of cell membrane, cell wall, and microtubule targets for antifungal action, antifungal development has been slow.
The polyene antifungals (e.g., amphotericin B and nystatin) target the fungal cell membrane, which is similar to mammalian plasma membranes, with the exception being that fungal plasma membranes contain ergosterol, rather than cholesterol as the principal sterol. The polyene amphotericin B remains the treatment mainstay for life-threatening and other mycoses, including candidiasis, cryptococcosis, aspergillosis, zygomycosis, coccidioidomycosis, histoplasmosis, blastomycosis, and paracoccidioidomycosis. Amphotericin B must be administered intravenously and is associated with numerous, often serious side effects, including phlebitis at the infusion site, fever, chills, hypokalemia, anemia and nephrotoxicity. Nystatin is another broad-spectrum polyene antifungal. However, its toxicity to humans prevents its widespread use. Currently, it is limited to topical applications, where it is effective against yeasts, including C. albicans. 
The azole antifungals (e.g., fluconazole, itraconazole, and ketoconazole) and the allylamine and morpholine antifungals (e.g., naftifine and terbinafine) interfere with ergosterol biosynthesis. Ketoconazole may be used to treat histoplasmosis, blastomycosis, mucosal candidiasis and various cutaneous mycoses (e.g., dermatophyte infections, pityriasis versicolor, and cutaneous candidiasis). However, it is not useful for treatment of aspergillosis or systemic yeast infections. Side effects associated with use of the azoles are not as severe as those associated with amphotericin B, although life-threatening hepatic toxicity may result from long-term azole use. Other side effects include nausea, vomiting, and drug interactions with such compounds as cyclosporin, antihistamines, anticoagulants, anti-seizure and oral hypoglycemic medications.
The few antimetabolite antifungals identified have not found widespread use. The most commonly used antifungal is 5-fluorocytosine, a fluorinated analog of cytosine. However, as with other antimetabolites, drug resistant fungal strains have emerged, and 5-fluorocytosine is seldom used alone. Nonetheless, in combination with amphotericin B, it remains the treatment of choice for cryptococcal meningitis, and is effective against some diseases caused by dematiaceous fungi.
Griseofulvin, an antifungal compound produced by Penicillium griseofulvin, acts by targeting microtubule-associated proteins. Griseofulvin is active against most dermatophytes, and is commonly used to treat dermatophytic infections. Potassium iodide is another compound that is used as an antifungal to enhance transepidermal elimination of fungal organisms in cases of cutaneous and lymphocutaneous sporotrichosis, although it is not effective against Sporothrix schenckii in vitro.
As with bacteria, drug-resistant strains of fungal pathogens have also been reported. This drug resistance can take various forms, such as primary resistance, where the susceptibility profiles for the species are characteristic, inherent, and rarely change in response to drug exposure, or the resistance can be secondary (i.e., acquired). Some of the molecular and cellular mechanisms by which fungal organisms acquire resistance are known (White, ASM News 63:427-433 [1997]; and White et al., Clin. Microbiol. Rev., 11:382-402 [1998]).
Significantly, fungal resistance to amphotericin B has been reported for various opportunistic fungi, including Pseudallescheria boydii, Fusarium, Trichosporon, and some C. lusitaniae and C. guilliermondii isolates (See, Dixon and Walsh, xe2x80x9cAntifungal Agents,xe2x80x9d in Baron (ed.), Medical Microbiology, 4th ed., University of Texas Medical Branch, Galveston, Tex. [1996], pp. 926-932). In addition, the emergence of azole-resistant fungal strains has raised concerns regarding use of the azole compounds, especially fluconazole, as a front-line treatment (Boschman et al., Antimicrob. Agents Chemother., 42:734 [1998]; Graybill, Clin. Infect. Dis., 22(Suppl. 2):S166 [1996]; White, ASM News 63:427-433 [1997]; and White et al., Clin. Microbiol. Rev., 11:382-402 [1998]).
In view of the development of resistance, as well as the relative lack of variety available in the selection of antifungals, there remains a need for the development of compounds useful for treatment of fungal diseases.
Selective Toxicity
The principle of selective toxicity is fundamental to the development of successful antimicrobial agents. That is to say, an antimicrobial compound, while toxic to the microorganism, ideally is not toxic to the subject receiving the antimicrobial compound. Selective toxicity is often a reflection of differences between the microorganism and host physiologies.
One approach to achieving selective toxicity is to identify a compound that is able to inhibit an essential enzyme in the microorganism, but due to differences in enzyme structure or function, that same antimicrobial compound does not affect the homologous enzyme in the host. Alternatively, an antimicrobial compound can inhibit a biochemical event that is essential to the microorganism, but that biochemical process may not be present or be essential to the host.
Chitin Biosynthesis
The fungal cell wall is essential for the viability of the organism, and is a rigid, stratified structure consisting of chitinous microfibrils and polysaccharides, among other components. The cell wall provides support and shape to the cell, and prevents osmotic lysis of the cell. Indeed, even a small lesion within the cell wall can lead to the extrusion of cytoplasm due to the positive intracellular pressure (See, Cole, xe2x80x9cBasic Biology of Fungi, in Baron (ed.), Medical Microbiology, 4th ed., University of Texas Medical Branch, Galveston, Tex. [1996], pp. 903-911). The yeast form of the C. albicans cell wall contains approximately 30-60% glucan, 25-50% mannan (mannoprotein), 1-2% chitin, 2-14% lipid, and 5-15% protein (McGinnis and Tyring, xe2x80x9cIntroduction to Mycology,xe2x80x9d in Baron (ed.), Medical Microbiology, 4th ed., University of Texas Medical Branch, Galveston, Tex. [1996], pp. 893-902). The chitin within the fungal cell wall is a (1-4)xcex2-linked polymer of N-acetyl glucosamine (GlcNAc) polymerized by chitin synthase at the plasma membrane (See, FIG. 1).
Chitin, although a minor component of yeast and filamentous fungal cell walls, is essential for cell viability and mother-daughter cell separation. Chitin biosynthesis, which requires uridine diphosphate-N-acetyl glucosamine (UDP-GlcNAc), is complex and is catalyzed by at least three gene products in S. cerevisiae, and perhaps as many as six gene products in certain filamentous molds (Bulawa, Ann. Rev. Microbiol., 47:505-534 [1993]; and Mellado et al., Mol. Genet., 246:353-359 [1995]). The three yeast genes, csI, csII and csIII, each have homologues in C. albicans and each performs a different intracellular function.
UDP-GlcNAc is the substrate for chitin synthase. Normal levels of UDP-GlcNAc are required for chitin biosynthesis and subsequent cell wall assembly and growth (Katz and Rosenberger, Biochim. Biophys. Acta., 208:452-460 [1970]). The pathway for the synthesis of UDP-GlcNAc, known as the Leloir pathway (Leloir and Cardini, Biochim. Biophys. Acta., 12:15-22 [1953]) is shown in FIG. 1. In this Figure, chitin synthase activity is shown for context, but is not considered part of the Leloir pathway. The first pathway-specific enzyme is 2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (known simply as ketol-isomerase; E.C. 5.3.1.19 or E.C. 2.6.1.16). The ketol-isomerase is an amino transferase that forms glucosamine-6-phosphate (GlcN-6-P) and glutamate from fructose-6-phosphate and glutamine (Selitrennikoff and Sonneborn, Develop. Biol., 54:37-51 [1976]). The second enzyme in the pathway is aminodeoxyglucosephosphate acetyltransferase (E.C. 2.4.1.4), which converts S-acetyl CoA and GlcN-6-P to CoA and N-acetylglucosamine-6-phosphate (GlcNAc-6-P) (Selitrennikoff and Sonneborn, Develop. Biol., 54:37-51 [1976]). The third enzyme is acetylaminodeoxyglucose phosphomutase (also known as GlcNAc-phosphomutase; E.C. 2.7.5.2) which converts GlcNAc-6-P to GlcNAc-1-phosphate, employing Glc-1,6-phosphate as a co-factor. The most downstream enzyme is UTP:acetylaminodeoxyglucose-1-phosphate uridylyl transferase (E.C. 2.7.7.23), which converts UTP and GlcNAc-1-phosphate to UDP-GlcNAc and pyrophosphate (PPi) (Selitrennikoff and Sonneborn, Biochim. Biophys. Acta., 451:408-416 [1976]; and Etchebehere et al., J. Bacteriol., 175:5022-5027 [1993]).
Chitin synthase has been a target for the identification of antifungal compounds for over 30 years, yet only two classes of compounds that target this enzyme have been identified. These are the competitive substrate inhibitors, namely the polyoxins and the nikkomycins. Each of these enzymatic inhibitors resembles the structure of the substrate, UDP-GlcNAc, and has inhibition constants (Ki) in the xcexcM range (Decker et al., J. Gen. Microbiol., 137:1805-1813 [1991]). Unfortunately, nikkomycin shows rapid degradation in biological fluids in rat, mouse and rabbit model systems (Tokumura and Horie, Biol. Pharm. Bull., 20:577-580 [1977]).
Thus, there remains a need to identify new antifungal compounds, and more specifically, there is a need to identify new classes of antifungal compounds that are effective against various fungal organisms, including those that are resistant to currently used compounds. Indeed, there remains a need to identify and develop new classes of antimicrobial compounds that are effective against multiple-drug resistant organisms. In addition, there is a need to identify and develop antimicrobial compounds that demonstrate selective toxicity towards microorganisms, but are not toxic, or have minimal (i.e., tolerable) toxicity, to animal hosts (e.g., humans).
The present invention provides methods for simultaneously assessing microbial phosphoglucose isomerase, ketol-isomerase and glucosamine-6-phosphate acetyltransferase activities, by measuring the production of Coenzyme A (CoA). The present invention finds use in the isolation of new classes of antifungal drugs, wherein the compounds have the ability to inhibit fungal glucose utilization.
In one embodiment, the present invention provides methods for the detection of phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities in a sample, comprising the steps of a) providing: a sample suspected to contain phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities, glucose-6-phosphate, glutamine, acetyl coenzyme A, and 5,5xe2x80x2-dithiobis(2-nitrobenzoic acid); b) combining the sample, glucose-6-phosphate, glutamine, and acetyl coenzyme A under conditions to yield reaction products comprising coenzyme A and N-acetylglucosamine-6-phosphate; c) inactivating the phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities; and d) combining the reaction product comprising coenzyme A and 5,5xe2x80x2-dithiobis(2-nitrobenzoic acid) under conditions to yield a chromogenic reaction product comprising 2-nitro-thiobezoate anion, wherein the chromogenic reaction product is indicative of phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities. In a related embodiment, the sample comprises a lysate selected from the group consisting of crude cell lysates and gel filtered cell lysates. In some embodiments, the lysate is a fungal cell lysate selected from the group consisting of Aspergillus cell lysates, Candida cell lysates, Cryptococcus cell lysates, Histoplasma cell lysates, Pneumocystis cell lysates, Rhizopus cell lysates, Saccharomyces cell lysates, and Schizosaccharomyces cell lysates. In one embodiment, the sample comprises purified fungal enzymes while in another embodiment the sample comprises recombinant fungal enzymes, where the fungal enzymes are selected from the group consisting of phosphoglucose isomerases, ketol-isomerases and acetyltransferases.
In other embodiments, the present invention provides methods for the detection of a compound having the ability to inhibit phosphoglucose isomerase, ketol-isomerase and/or acetyltransferase activities in a sample, comprising the steps of: a) providing a sample suspected to contain phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities, glucose-6-phosphate, glutamine, acetyl coenzyme A, 5,5xe2x80x2-dithiobis(2-nitrobenzoic acid), and a candidate compound; b) preparing a first and second reaction mixture, where the first reaction mixture comprises the sample, glucose-6-phosphate, glutamine, and acetyl coenzyme A, and where the second reaction mixture comprises the sample, glucose-6-phosphate, glutamine, acetyl coenzyme A and the candidate compound; c) incubating the first and second reaction mixtures under conditions to yield reaction products comprising coenzyme A and N-acetylglucosamine-6-phosphate; d) inactivating the phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities; e) combining the first and second reaction mixtures with 5,5xe2x80x2-dithiobis(2-nitrobenzoic acid) under conditions to yield a chromogenic reaction product comprising 2-nitro-thiobezoate anion; and f) comparing the quantity of the chromogenic reaction product in the first and second reaction mixtures. In a related embodiment, these methods further comprise step g) scoring the candidate compounds as positive for the ability to inhibit phosphoglucose isomerase, ketol-isomerase and/or acetyltransferase activities in a sample, when the second reaction mixture yields less than 50% of the chromogenic reaction product of the first reaction mixture. In other embodiments, the sample comprises a lysate selected from the group consisting of crude cell lysates and gel filtered cell lysates. In some embodiments, the lysate is a fungal cell lysate selected from the group consisting of Aspergillus cell lysates, Candida cell lysates, Cryptococcus cell lysates, Histoplasma cell lysates, Pneumocystis cell lysates, Rhizopus cell lysates, Saccharomyces cell lysates, and Schizosaccharomyces cell lysates. In one embodiment, the sample comprises purified fungal enzymes, while in another embodiment the sample comprises recombinant fungal enzymes, where the fungal enzymes are selected from the group consisting of phosphoglucose isomerases, ketol-isomerases and acetyltransferases. In some embodiments, the candidate compound is present in an extract selected from the group consisting of extremophile extracts, marine macroorganism extracts, cyanobacterial extracts and algal extracts.
In some embodiments, the present invention also provides compositions comprising at least one candidate compound which has the ability to inhibit phosphoglucose isomerase, ketol-isomerase and/or acetyltransferase activities in a sample, identified by methods comprising the steps of: a) providing a sample suspected to contain phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities, glucose-6-phosphate, glutamine, acetyl coenzyme A, 5,5xe2x80x2-dithiobis(2-nitrobenzoic acid), and a candidate compound; b) preparing a first and second reaction mixture, where the first reaction mixture comprises the sample, glucose-6-phosphate, glutamine, and acetyl coenzyme A, and where the second reaction mixture comprises the sample, glucose-6-phosphate, glutamine, acetyl coenzyme A and the candidate compound; c) incubating the first and second reaction mixtures under conditions to yield reaction products comprising coenzyme A and N-acetylglucosamine-6-phosphate; d) inactivating the phosphoglucose isomerase, ketol-isomerase and acetyltransferase activities; e) combining the first and second reaction mixtures with 5,5xe2x80x2-dithiobis(2-nitrobenzoic acid) under conditions to yield a chromogenic reaction product comprising 2-nitro-thiobezoate anion; and f) comparing the quantity of the chromogenic reaction product in the first and second reaction mixtures. In a related embodiment, these compositions comprising at least one candidate compound which has the ability to inhibit phosphoglucose isomerase, ketol-isomerase and/or acetyltransferase activities in a sample are identified by methods which further comprise step g) scoring the candidate compounds as positive for the ability to inhibit phosphoglucose isomerase, ketol-isomerase and/or acetyltransferase activities in a sample, when the second reaction mixture yields less than 50% of the chromogenic reaction product of the first reaction mixture. In some embodiments, the candidate compound is present in an extract selected from the group consisting of extremophile extracts, marine macroorganism extracts, cyanobacterial extracts and algal extracts. In other embodiments, the candidate compound is present in a high performance liquid chromatography (HPLC) fraction of a microbial extract. In one embodiment, the candidate compound further has antifungal activity. In some embodiments, the antifungal activity is determined by a test selected from the group consisting of agar diffusion assays, broth dilution assays, and animal model assays. In preferred embodiments, the antifungal activity is selected from the group consisting of anti-Aspergillus activity, anti-Candida activity, anti-Cryptococcus activity, anti-Histoplasma activity, anti-Pneumocystis activity, anti-Rhizopus activity, anti-Saccharomyces activity, and anti-Schizosaccharomyces activity. In some embodiments the candidate compound further has limited toxicity to mammalian cells. In preferred embodiments, the mammalian cells are selected from the group consisting of murine cells and human cells. In a further preferred embodiment, the limited toxicity is determined by a test selected from the group consisting of in vitro and in vivo acute toxicity tests.